Rhodamine dyes are known to be photostable fluorescent labels with large absorption coefficients, high fluorescent quantum yields, and low degree of triplet formation. Rhodamines are widely used both as laser dyes and fluorescent compounds for labelling proteins, nucleic acids, lipids, carbohydrates, toxins, hormones and other biomolecules (e.g. R. P. Haugland, A Guide to Fluorescent Probes and Labelling Technologies, Invitrogen, Carlsbad, 2005, pp. 11-37).
Rhodamines have also served for practical implementations of some new physical concepts that overcome the diffraction limit by switching between the dark and bright states of a fluorescent marker (e.g. S. W. Hell, Science 2007, 317, 1749-1753). For example, a very important novel method of the stimulated emission depletion (STED) microscopy uses the ground (singlet) state of the fluorophore (S0) as a dark state, and the first excited state (S1) as a bright one. In practical applications of the STED method, a focused pulse excites fluorescence in a small spot (with dimensions limited by diffraction), and immediately after that a red-shifted doughnut-shaped STED beam switches off the fluorescence of excited molecules by stimulated emission (S1→S0) everywhere, except in the very center of the doughnut, where the quenching intensity is zero. For squeezing the fluorescence to a very small central spot, the depletion rate should exceed the rate of the spontaneous transition to a ground state S0. Fluorescent lifetimes of organic fluorophores (τfl˜10−9 s) and their optical cross-sections of the S1→S0 transitions (σ˜10−16 cm2) imply that the STED-pulse should have a very high power ISTED>>IS≡(στfl)−1˜1025 photons/(cm2×s)≅10 MW/cm2. IS is a threshold intensity depending on the dye employed and the depletion wavelength used. The resolution enhancement scales roughly with √{square root over (1+ISTED/IS)}. These huge light intensities inevitably cause photobleaching of fluorophores, and therefore STED microscopy ultimately requires the most photostable fluorescent dyes.
Along with the relatively long lifetimes of the excited states (>3 ns), other important qualities of the STED and common fluorescent dyes are high fluorescent quantum yields (Φfl) and oscillator strengths (high absorption coefficients), low rate of the triplet state formation, sufficient solubility in water or aqueous buffers and a reactive group (with a linker) for attaching a dye to a biological object or any other structure of interest. High Φfl-values of the fluorescent labels conjugated with biomolecules are very important, as they improve the sensitivity of the imaging method. Moreover, if a resolution on the molecular scale is desired, or if only single molecules remain in the effective detection volume, the fluorophores should be suitable for the single molecule detection (e.g. in the method of fluorescence correlation spectroscopy—FCS). Recently, a far-field fluorescence “nanoscopy” based on switching the majority of the fluorescent molecules to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state, has been introduced (S. W. Hell et. al, Nature Meth. 2008, 5, 943-945). This superresolution imaging method of the ground state depletion and single molecule return (GSDIM) requires new photostable fluorescent dyes with recovery times from several tens to several hundreds of milliseconds, minimal content of the dye in the ground state after the pump pulse and the possibility of the enhanced recovery caused by the irradiation with the UV laser (375 nm).
Water is the preferred solvent for operating with the reactive fluorescent dyes, because the conjugation reactions involving biologically relevant macromolecules (proteins, nucleic acids, carbohydrates) need to be performed in water or aqueous buffers. A marker is usually dissolved in an organic solvent, such as DMF or DMSO, and then added to the aqueous solution of the substrate. High concentrations of an organic solvent may cause protein denaturation, and hence should be avoided. On the other hand, a low coupling efficiency may be observed if the amount of the organic solvent is too low, and the marker precipitates in the reaction mixture. Water-soluble fluorescent markers are advantageous in this regard, because they do not require any organic solvents at all. Moreover, hydrophilic labels are less prone to aggregation and to non-specific binding with biological objects, especially membranes. Another advantageous feature of a potential fluorescent label is the availability of the two forms of the same dye (chromophore)—a lipophilic and hydrophilic one. The latter is indispensable for labelling of non-polar compounds (e.g. lipids in their non-polar domain), and the former is irreplaceable for labelling of polar substances (e.g. lipid head-groups).
Biological applications require fluorescent dyes absorbing in the red spectral region, because the excitation in this area reduces the background originating from autofluorescence of the cells (evident with UV and blue excitation). Very convenient is the excitation by the red He—Ne laser at 633 nm or with the 635 nm spectral line of a red diode laser, as well as with the 647 nm line of the krypton ion laser or with a diode laser emitting at 650 nm. Many fluorescent dyes have been prepared for these excitation sources. Various types of the commercially available dyes are given in Table 1. The lack of the “ideal” fluorescent dye for operating in the “red” spectral region becomes clear when one compares the performance of the available dyes under STED and FCS conditions in microscopy with very high light intensities and in the presence of air-oxygen.
TABLE 1Commercially available organic fluorescent dyes for the excitation with He—Nered laser (633 nm) or the 635 nm spectral line of red diode lasers.*λmaxλmaxε · 10−5(abs.)(fl.)Sol-M−1ΦflτflProviderSolubilityNamenmnmventcm−1%ns(structure)(polarity)BODIPY ®625640MeOH1.01“good”3.9 (H2O)InvitrogenDMSO, MeCN630/6504.4 (EtOH)1(+)(non-polar)Atto 6332629657H2O1.3643.2 (H2O)Atto-tecDMF, DMSO,(−)(H2O) MeCN2Alexa ®632647MeOH1.0—3.2InvitrogenDMSO, H2O36333(+)Atto 635635659H2O1.2251.9Atto-tecDMF, DMSO,(+)MeCN (H2O)2,4Atto 6375635659H2O1.2251.9Atto-tecH2O, DMF,(−)DMSO,MeCN,4,5DyLight ®638658un-2.0——Thermo—633knownFisher Sci.(−)DY-6306636657EtOH2.0Varies60.21DyomicsMeOH, EtOH,6271 6511H2O(H2O)1,6(+)DMF, DMSODY-6317,635-657-EtOH2.0——DyomicsH2O, MeOH,6327,637658(+)DMF, DMSO6337, 6347DY-635647671EtOH2.0—0.48DyomicsMeOH, EtOH, 6351 6691H2O(H2O)1,6(+)DMF, DMSODY-6368645671EtOH2.0——DyomicsH2O, EtOH,(+)DMF, DMSODY-6509653674EtOH2.20.64 (H2O)1DyomicsMeOH, EtOH, 6461670H2O(+)DMF, DMSOEvoblue647664EtOH1.00.64 (H2O)1FlukaH2O, MeOH,309 6501 6671H2O(+)DMF, DMSOCy ® 59647663H2O2.5271 (PBS)11GE Health-DMF, DMSO(PBS)100.9 (H2O)1care(H2O)(+)Alexa ®651672MeOH2.7 33141.0 (H2O)1InvitrogenH2O, DMSO6479,12,13 6491 6661H2O(+)15hydrophilicAtto644669PBS111.5653.4Atto-tecDMF, DMSO647N9,16,17(+)18(non-polar)2DyLight ®646674—2.5——Thermo—6499Fisher Sci.(−)*λmax (abs.), λmax (fl.): absorption and fluorescence maxima, respectively; ε: molar extinction coefficient; τfl: excited state lifetime.1V. Buschmann, K. D. Weston, M. Sauer, Bioconj. Chem. 2003, 14, 195-204.2According to the definition of the producer, this dye is “moderately hydrophilic”.3B. Agnew, K. R. Gee, T. G. Nyberg (Invitrogen), U.S. Pat. U.S. 2007/02490144Slowly decomposes at pH > 8.5.5Hydrophilic version of Atto 635.6Addition of (bio)polymers (BSA, Tween20) increases the low fluorescence quantum yield of DY fluorophores (ca. 5% in H2O) and their fluorescence lifetime: P. Czerney, F. Lehmann, M. Wenzel, V. Buschmann, A. Dietrich, G. J. Mohr, Biol. Chem. 2001, 382, 495-4987The same fluorophore as in DY-630; the solubility in water is increased due to the presence of up to 4 sulfonic acid residues.8The same fluorophore as in DY-635; the solubility in water is increased due to the presence of two sulfonic acid residues.9Fluorescence of this dye may be excited by the 647 nm line of the krypton ion laser or with diode laser emitting at 650 nm.10R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis, A. S. Waggoner, Bioconj. Chem. 1993, 4, 105-111.11www.iss.com/resources/fluorophores.html (PBS = phosphate buffer saline: 50 mM potassium phosphate, 150 mM NaCl, pH 7.2)12Cy ® 5 dye was shown to be “brighter” but less photostable than Alexa ® 647: J. L. Ballard, V. K. Peeva, C. J. de Silva, J. L. Lynch, N. R. Swanson, Mol. Biotechnol. 2007, 36, 175-183.13Photostability of the fluorescent dyes decreases in the following order: Alexa ® 647 > Alexa ® 633 > Cy ® 5: R. P. Haugland, A Guide to Fluorescent Probes and Labelling Technologies, Invitrogen Corp., Carlsbad, 2005, p. 38.14Measured at 22° C. relative to fluorescein in 0.01M NaOH (Φfl = 0.92); www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/tables/Fluorescence-quantum-yields-and-lifetimes-for-Alexa-Fluor-dyes.html15For the structure of Alexa 647, see Supporting Information of the following report: M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, Science, 2007, 317, 1749-1753.16According to the data of Atto-tec GmbH, photoresistance of the fluorescent dyes decreases in the following order: Atto 633 > Atto 647 > Cy ® 5: www.atto-tec.de17Mixture of two diastereomers with practically identical physical properties.18For the structure of Atto 647N, see Supporting Information of the following report: C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. v. Middendorff, A. Schönle, S. W. Hell, Nature, 2009, 457, 1159-1163.
Analysis of the disclosed structures of the commercial fluorescent dyes matching the excitation with He—Ne red laser (633 nm) or 635 nm spectral line of red diode lasers (Tab. 1, FIG. 1) reveals that there is only one rhodamine among them: Alexa 633 was reported to be a “sulfonated rhodamine derivative” [J. E. Berlier et al., J. Histochem. Cytochem. 2003, 51, 1699-1712], and only in 2007 its structure has been reported [B. Agnew, K. R. Gee, T. G. Nyberg (Invitrogen), US Pat. 2007/0249014]. Along with rhodamines, the following substance classes were considered as leads for developing of the new fluorescent dyes: BODIPY® derivatives (represented by BODYPY® 630/650 in FIG. 4), carbopyronines (Atto 635 and 647), cyanine dyes (Cy® 5 and Alexa 647), structurally related hybrids of cyanine dyes and benzopyranes (DY-630/635/650) and oxazines (Evoblue 30). BODIPY® derivatives have a substantial drawback: sometimes, fluorescence intensity of their bioconjugates is not directly proportional to a number of labelled sites [R. P. Haugland, A Guide to Fluorescent Probes and Labelling Technologies, Invitrogen Corp., Carlsbad, 2005, p. 53]. Moreover, it is difficult to chemically modify a BODIPY residue in such a way that it becomes water-soluble and more photostable. Carbopyronines have been extensively studied by Drexhage et al. [e.g. A. Zilles, J. Arden-Jacob, K.-H. Drexhage, N. U. Kemnitzer, M. Hammers-Schneider (Atto-tec GmbH), WO 2005/003086 (13 Jan. 2005)]. The carbopyronine dye Atto 647N has been widely used for labelling in many “nanoscopic” studies. Quite recently first STED images of the living cells were recorded at a rate of 28 frames per seconds using this dye [V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, S. W. Hell, Science 2008, 320, 246-249]. In the course of this study, the movements of synaptic vesicles inside the axons of cultured neurons were recorded with a resolution of ca. 60 nm. Due to the low polarity of the dye, it sticks to the walls of micro capillary injection tubes, so that it proved difficult to properly inject its solutions into the cell. Sometimes bioconjugates of Atto 647N with antibodies display a strong increase in intensity of the second absorption peak at about 605 nm (irradiation at this wavelength does not generate any emission). Moreover, the lipophilic Atto 647N produced a considerable background in immunostaining experiments, largely due to its affinity to mitochondria. However, it is not easy to further improve the properties of the carbopyronine dye Atto 647N and make it more hydrophilic. Photostability of this dye was found to be better than that of the spectrally similar Alexa® 647. Therefore, the following order of the photoresistance can be derived: Atto 633>Atto 647N>Alexa® 647>Alexa® 633>Cy® 5 (see ref. to Table 1). Lower photostability and moderate Φfl-values of cyanine dyes make them inappropriate as lead structures. Short lifetimes of the excited states and presumably low Φfl-values of oxazines (e.g. Evoblue 30), as well as their moderate photostability, make the optimization of their properties not very promising.
Though rhodamines have been known and studied for a very long time, further improvements of their properties are still possible. For example, a very large bathochromic shift has been achieved for rhodamine 700 with a skeleton of the well-known rhodamine 101, in which the benzoic acid residue is replaced by trifluoromethyl group. All rhodamine derivatives with a perfluoroalkyl group at the position 9 were found to absorb and emit above 600 nm [M. Sauer et al., J. Fluoresc. 1995, 5, 247-261]. Unfortunately, they cannot be used as scaffolds for derivatization and further improvements, because the presence of the small and very strong electron acceptor group at C-9 of the xanthene fragment (“opposite” the oxygen atom) makes this position very vulnerable to the nucleophilic attack by water. Therefore, such rhodamines decolorize rapidly in aqueous solutions and loose their fluorescence. Up to now, the highest values for the adsorption and emission maxima (630 and 655 nm, respectively) for “normal” rhodamines have been achieved for the rigidized xanthene derivative 4 (Scheme 1) in 8 M urea solution [L. G. Lee, R. J. Graham, W. E. Werner, E. Swartzman, L. Lu, (Apptera Corp., USA), U.S. Pat. No. 6,372,907 (16 Apr. 2002)]. The drawback of compound 4 is its high lipophilicity (low polarity) and therefore low solubility in water or aqueous buffers. Another drawback of this compound is that it has a free carboxylic group which may give a colourless and non-fluorescent cyclic ester form. Though compound 4 is a valuable intermediate, it lacks any suitable reactive site for attaching to biomolecules. The carboxylic group in compound 4 is sterically hindered and low reactive. Moreover, the reaction of this carboxylic acid with primary amino groups (e.g. in proteins, lipids, etc.) would give amides which are known to form colourless and non-fluorescent cyclic spiroamides (due to addition of NH-group across the double tetrasubstituted C9═C8a/8b bond in the central xanthene ring).
Similar spectral values (624 and 644 nm for the absorption and emission, respectively) have been recorded in ethanol for the ethyl esters of the tetrachloro rhodamines AZ 84-AZ 95 [compounds 71-82 in WO 2005/003086].
In view of the drawbacks of the fluorescent dyes of the prior art, the main object of the present invention was to provide novel fluorescent dyes which would exhibit improved properties, namely photostability in aqueous solutions, hydrophilicity, high values for adsorption and emission maxima, recovery times of several tens—several hundreds of milliseconds, minimal content of the dye in the ground state after the pump pulse, the possibility of the enhanced recovery caused by the irradiation with the UV light, and which would be particularly suitable for microscopy applications with very high light intensities such as STED, FCS and GSDIM.
This object has been achieved by providing the novel rhodamines according to the invention.